Cytochrome c oxidase is one of the key energy-generating proteins that function to pump protons across the inner membrane of mitochondria. The catalytic core of the enzyme is composed of three integral membrane proteins, subunits I, II and III. Subunit III does not play a direct role in electron transfer, but it does affect the function of the proton transfer pathways and the proton pumping mechanism in subunit I. Subunit III also prevents the oxidase from undergoing rapid suicide inactivation during normal catalytic turnover. High resolution structures of cytochrome oxidase show that subunit III is associated with tightly bound phospholipids. Mutagenesis and phospholipase treatment of cytochrome c oxidase of the bacterium Rhodobacter sphaeroides, which is closely related to the mitochondrial oxidase, indicates that these lipids are necessary for binding subunit III to subunit I. This is explained by the oxidase structures which show that four phospholipids are simultaneously coordinated by conserved residues of both subunit I and subunit III. The first aim of this project is to examine the protein-lipid interactions of cytochrome oxidase through the analysis of site-directed mutants designed to interrupt these interactions. These experiments will yield information about how tightly bound phospholipids function to facilitate the binding of membrane proteins. These studies should also reveal what type of inter-subunit interactions between integral membrane proteins are used to transmit structural information. A second aim is to study the process of long-distance proton transfer in cytochrome oxidase, using site-directed mutagenesis of subunit III and subunit I residues. Key questions here are how the protein surface around the entry site of a proton pathway controls the rate of proton transfer and how sensitive is the proton conductive pathway in the interior of the protein to modification of residues that connect the pathway to another subunit. A third aim is to establish what structural changes occur with suicide inactivation in order to understand the mechanism of the inactivation process. A fourth aim is to characterize a copper chaperone that appears to be involved in the insertion of CuB into heme a3-CuB active site of cytochrome oxidase. The mechanism of this assembly process is likely to differ from the assembly of other copper centers in proteins since the active site of cytochrome oxidase is buried within subunit I and within the transmembrane region of the protein. [unreadable] [unreadable]